Vaccinia virus is the focus of much attention as a vector for expression systems, gene delivery, vaccination, and as a model for viral pathogenesis. However, a major limitation in the ability to utilize vaccinia vectors are concerns about pathogenicity. The study of how vaccinia spreads and evades the host immune response as well as ways to enhance the immunogenicity of expressed antigens, is thus essential for the effective utilization of this vector system. The vaccinia B5R gene encodes a 42-kDa protein found on the membranes of extracellular virions and infected cells. In preliminary studies, we have shown that B5R plays a critical role in cell-to-cell spread and long range dissemination of the virus through the formation of the extracellular enveloped virus (EEV). In addition, the B5R extracellular domain contains repeat units, homologous to complement regulatory proteins, which may be involved in viral evasion of host immune responses. However, we showed that both this domain and the cytoplasmic tail are dispensable for EEV formation and therefore may be utilized as sites for heterologous antigen expression. We hypothesize that B5R is a multifunctional protein that plays a central role in vaccinia pathogenesis, dissemination, and evasion of host immune responses; that by modification of B5R, these in vivo properties of vaccinia can be altered; and that B5R can be manipulated to traffic heterologous antigens to EEV resulting in vaccinia vectors that generate enhanced immunity. The aim of this study is to better understand the multifunctional B5R protein in vaccinia pathogenesis and exploit it for heterologous protein expression in order to create vaccinia-based vaccines with novel biological and antigen presentation capabilities. To do this we will: 1) define the functional significance of B5R domains for vaccinia pathogenesis in vivo; 2) identify the complement regulatory properties of B5R; and 3) Determine the immune responses generated to novel vaccinia vaccines engineered to express an HIV antigen fused to B5R. We anticipate that these studies will give insight into vaccinia pathogenesis and the role of B5R in dissemination and evasion of host immune responses; provide a mechanism of modulating the pathogenicity of vaccinia vectors; and offer unique approaches to vaccinia-based antigen expression that can be exploited for novel vaccine strategies.